EP3359640A2 - Methods of estimating logd of tagged combinatorial library compounds - Google Patents
Methods of estimating logd of tagged combinatorial library compoundsInfo
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- EP3359640A2 EP3359640A2 EP16854552.3A EP16854552A EP3359640A2 EP 3359640 A2 EP3359640 A2 EP 3359640A2 EP 16854552 A EP16854552 A EP 16854552A EP 3359640 A2 EP3359640 A2 EP 3359640A2
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- logd
- ligand
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Definitions
- the methods generally involve contacting the compounds with a matrix, measuring the interaction of the compounds with the matrix and relating the interaction of the compounds with the matrix to LogD of the compounds.
- novel compounds which include a ligand operatively linked to a recognition element and linkers that connect the ligand to the recognition element.
- Combinatorial libraries which were first developed over thirty years ago, now routinely identify novel, high affinity ligands for wide variety of biological targets (e.g., receptors, enzymes, nucleic acids, etc.) and hence are of increasing importance in drug discovery.
- Tagged combinatorial libraries particularly libraries which use DNA as a tag to record the synthetic steps undergone by ligands operatively attached to the DNA, are of particular current interest.
- Advances in DNA sequencing, PCR technology and ligand assay development provide methods to identify and select ligands operatively linked to DNA that bind to a biological target, from complex mixtures of ligands operatively linked to DNA (Harbury, et al., U.S. Patent No. 7,479,472; Liu et al., U.S Patent No. 7,070,928; Liu et al., U.S Patent No. 7,223,545; Liu et al., U.S. Patent No. 7,442,160; Liu et al., U.S. Patent No. 7,491,160; Liu et al., U.S. Patent No.
- Patent No. 8,722,583 Freskgard et al, U.S. Patent Application No.
- lipophilicity can be routinely measured for individual organic compounds
- methods for estimating lipophilicity for ligands operatively linked to recognition elements in a complex mixture of similar ligands such as those provided by tagged combinatorial chemistry methods have not yet been developed. Accordingly, what is needed are methods for estimating lipophilicity of members of combinatorial libraries, where the ligands are operatively linked with recognition elements. Such methods will greatly assist in identifying compounds derived from combinatorial libraries with properties amenable to further optimization as drug candidates and accordingly, increase the efficiency of drug development.
- a method of estimating LogD of one compound which includes a ligand operatively linked to a recognition element includes contacting the compound with a lipid matrix, separating the compound absorbed by the lipid matrix from the compound not absorbed by the lipid matrix, measuring the amount of the compound absorbed by the lipid matrix and/or the amounts of the compound not absorbed by the lipid matrix where measurement of the amount of the compound absorbed by the lipid matrix and/or the amounts of the compound not absorbed by the lipid matrix provide an estimation of LogD of the compound.
- a method of estimating LogD of two or more compounds which include a ligand operatively linked to a recognition element includes contacting the compounds with a lipid matrix, separating compounds absorbed by the lipid matrix from compounds not absorbed by the lipid matrix, measuring the amounts of compounds absorbed by the lipid matrix and/or the amounts of compounds not absorbed by the lipid matrix where measurement of the amounts of compounds absorbed by the lipid matrix and/or the amounts of the compounds not absorbed by the lipid matrix provide an estimation of LogD of the compounds.
- a method of estimating LogD of one compound which includes a ligand operatively linked to a recognition element comprises contacting the compound with a gel matrix, applying a voltage gradient to the gel matrix and measuring the Rf of the compound on the gel matrix wherein the Rf of the compound on the gel matrix provides an estimate of LogD of the compound.
- a method of estimating LogD of two or more compounds which include a ligand operatively linked to a recognition element is provided. The method includes the steps of contacting the compounds with a gel matrix, applying a voltage gradient to the gel matrix and measuring the R f of the compounds on the gel matrix wherein the R f of the compounds on the gel matrix provides an estimate of LogD of the compounds.
- a method of estimating LogD of one compound which include a ligand operatively linked to a recognition element includes the steps of contacting the compound with a chromatographic matrix and measuring the retention times of the compound on the
- a method of estimating LogD of two or more compounds which include a ligand operatively linked to a recognition element includes the steps of contacting the compounds with a chromatographic matrix and measuring the retention times of the compounds on the chromatographic matrix wherein the measured retention times of the compounds on the chromatographic matrix provides an estimate of LogD of the compounds.
- a double stranded DNA molecule comprising one oligonucleotide operatively linked to a variable first ligand at the 3' terminus hybridized to a complementary oligonucleotide operatively linked to a constant second ligand at the adjacent 5' terminus wherein the LogD of the second ligand is greater than 6 is provided.
- above molecule is used in a method of estimating LogD of two or more first ligands of double stranded DNA molecules.
- the method includes the steps of contacting the double stranded DNA molecules with a lipid matrix, separating the double stranded DNA molecules absorbed by the lipid matrix from double stranded DNA molecules not absorbed by the lipid matrix and measuring the amounts of double stranded DNA molecules absorbed by the lipid matrix and/or the amounts of double stranded DNA molecules not absorbed by the matrix wherein measurement of the amounts of double stranded DNA molecules absorbed by the matrix and/or the amounts of the double stranded DNA molecules not absorbed by the matrix provide an estimate of LogD of the first ligands of the double stranded DNA molecules.
- RE C is a recognition element
- Lb is a ligand with similar or identical hydrophobicity
- C3 and C 4 are independently linkers
- X 4 , X5 and X 6 are functional groups and k and 1 or independently 0 or 1.
- a compound comprising a compound of Formula (I) hybridized to a compound of Formula (II) wherein RE and RE C are independently an oligonucleotide, single stranded RNA or single stranded DNA, Ci, C 2 , n, o, Xi, X 2 , X3, L a , Lb C3, C 4 , k, 1, X 4 , X5 and X 6 are as defined above is provided.
- Figure 1 illustrates that compounds 1 and 2 attached to the 5 ' end of DNA molecules are modestly retarded on a standard agarose gel.
- Figure 2 illustrates that compounds 1 and 2 attached to the 5 ' end of DNA molecules are retarded on a standard agarose gel which includes 0.9% (w/v) synthetic vesicles.
- FIG. 3 is a HPLC trace which illustrates that compounds 4-7 were resolved by reversed phase HPLC.
- Figure 4 illustrates the recovered area for compounds 2-7 after mixing with a matrix.
- Figure 5 illustrates the recovered area for compounds 8-11 after mixing with a matrix.
- Figure 6 illustrates the recovered area for compounds 13-17 after mixing with a matrix.
- Figure 7 illustrates the structure of the amine linker and pyrrolo dC.
- Figure 8 illustrates analytical data for an exemplary bile acid conjugate.
- Figure 9 is a HPLC trace which illustrates that compounds 19-24 were resolved by reversed phase HPLC.
- Figure 10 illustrates the retention time of bile acids conjugated to 220 base pair oligonucleotide plotted against cLogP of the bile acid methyl amides
- Figure 11 illustrates the retention time of the peptides of Example 12 plotted against their measured eLogD values.
- Figure 12 illustrates the retention time of the peptide oligomers of Example 13 plotted against eLogD values of the free peptides.
- the terms “about” and “approximately,” when used in connection with a property with a numeric value or range of values indicate that the value or range of values may deviate to an extent deemed reasonable to one of ordinary skill in the art while still describing the particular property. Specifically, the terms “about” and “approximately,” when used in this context, indicate that the numeric value or range of values may vary by 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1 % of the recited value or range of values while still describing the particular solid form.
- Alkyl by itself or as part of another substituent, refers to a saturated or unsaturated, branched, straight-chain or cyclic monovalent hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene or alkyne.
- Typical alkyl groups include, but are not limited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-l-yl, propan-2-yl, cyclopropan-l-yl, prop-l-en-l-yl, prop-l-en-2-yl, prop-2-en-l-yl (allyl), cycloprop-l-en-l-yl; cycloprop-2-en-l-yl, prop-l-yn-l-yl, prop-2-yn-l-yl, etc. ; butyls such as butan-l-yl, butan-2-yl, 2-methyl-propan-l-yl,
- alkyl is specifically intended to include groups having any degree or level of saturation, i.e. , groups having exclusively single carbon-carbon bonds, groups having one or more double carbon-carbon bonds, groups having one or more triple carbon-carbon bonds and groups having mixtures of single, double and triple carbon-carbon bonds. Where a specific level of saturation is intended, the expressions "alkanyl,” “alkenyl,” and “alkynyl” are used.
- an alkyl group comprises from 1 to 20 carbon atoms (C1-C20 alkyl). In other embodiments, an alkyl group comprises from 1 to 10 carbon atoms (C1-C10 alkyl). In still other embodiments, an alkyl group comprises from 1 to 6 carbon atoms (Ci-C 6 alkyl).
- Alkanyl by itself or as part of another substituent, refers to a saturated branched, straight-chain or cyclic alkyl radical derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane.
- Typical alkanyl groups include, but are not limited to, methanyl; ethanyl; propanyls such as propan-l-yl, propan-2-yl (isopropyl), cyclopropan-l-yl, etc. ; butanyls such as butan-l-yl, butan-2-yl (seobutyl), 2-methyl-propan-l-yl (isobutyl),
- alkenyl by itself or as part of another substituent, refers to an unsaturated branched, straight-chain or cyclic alkyl radical having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene. The group may be in either the cis or trans conformation about the double bond(s).
- Typical alkenyl groups include, but are not limited to, ethenyl; propenyls such as prop-l-en-l-yl, prop-l-en-2-yl, prop-2-en-l-yl (ally 1) , prop-2-en-2-yl, cycloprop-l-en-l-yl; cycloprop-2-en-l-yl; butenyls such as but-l-en-l-yl, but-l-en-2-yl, 2-methyl-prop-l-en-l-yl, but-2-en-l-yl , but-2-en-l-yl, but-2-en-2-yl, buta-l,3-dien-l-yl,
- Alkynyl by itself or as part of another substituent refers to an unsaturated branched, straight-chain or cyclic alkyl radical having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne.
- Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop-l-yn-l-yl, prop-2-yn-l-yl, etc. ; butynyls such as but-l-yn-l-yl, but-l-yn-3-yl, but-3-yn-l-yl, etc. ; and the like.
- Alkyldiyl by itself or as part of another substituent, refers to a saturated or unsaturated, branched, straight-chain or cyclic divalent hydrocarbon group derived by the removal of one hydrogen atom from each of two different carbon atoms of a parent alkane, alkene or alkyne, or by the removal of two hydrogen atoms from a single carbon atom of a parent alkane, alkene or alkyne.
- the two monovalent radical centers or each valency of the divalent radical center can form bonds with the same or different atoms.
- Typical alkyldiyl groups include, but are not limited to methandiyl; ethyldiyls such as ethan-l,l-diyl, ethan-l,2-diyl, ethen-l,l-diyl, ethen-l,2-diyl; propyldiyls such as propan-l,l-diyl,
- alkyldiyl group is (C1-C20) alkyldiyl. In other embodiments, the alkyldiyl group is (C1-C10) alkyldiyl. In still other
- the alkyldiyl group is (Ci-C 6 ) alkyldiyl.
- saturated acyclic alkanyldiyl groups in which the radical centers are at the terminal carbons e.g. , methandiyl (methano); ethan-l,2-diyl (ethano);
- propan- 1,3-diyl propano
- butan-l,4-diyl butano
- alkyleno also referred to as alkyleno, defined infra
- Alkyleno by itself or as part of another substituent, refers to a straight-chain alkyldiyl group having two terminal monovalent radical centers derived by the removal of one hydrogen atom from each of the two terminal carbon atoms of straight-chain parent alkane, alkene or alkyne.
- Typical alkyleno groups include, but are not limited to, methano; ethylenos such as ethano, etheno, ethyno; propylenos such as propano, prop[l]eno, propa[l,2]dieno, prop[l] yno, etc.; butylenos such as butano, but[l]eno, but[2]eno, buta[l,3]dieno, but[l]yno, but[2]yno, but[l,3]diyno, etc. ; and the like. Where specific levels of saturation are intended, the nomenclature alkano, alkeno and/or alkyno is used.
- the alkyleno group is (C1-C20) alkyleno. In other embodiments, the alkyleno group is (C1-C10) alkyleno. In still other embodiments, the alkyleno group is (Ci-C 6 ) alkyleno. In some embodiments, straight-chain saturated alkano groups, e.g. , methano, ethano, propano, butano, and the like are preferred.
- Antibody refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes, e.g. , a fragment containing one or more complementarity determining region (CDR).
- the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
- Light chains are typically classified as either, e.g., kappa or lambda.
- Heavy chains are typically classified e.g.
- a typical immunoglobulin (antibody) structural unit comprises a tetramer.
- each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy" chain (about 50-70 kD).
- the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
- the terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
- Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
- pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2 (fragment antigen binding) and Fc (fragment crystallizable, or fragment complement binding).
- F(ab)'2 is a dimer of Fab, which itself is a light chain joined to VH-CH1 by a disulfide bond.
- the F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab') 2 dimer into a Fab' monomer.
- the Fab' monomer is essentially a Fab with part of the hinge region.
- the Fc portion of the antibody molecule corresponds largely to the constant region of the immunoglobulin heavy chain, and is responsible for the antibody's effector function (see, Fundamental Immunology, 4 th edition. W.E. Paul, ed., Raven Press, N.Y. (1998), for a more detailed description of antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' or Fc fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology, peptide display, or the like.
- antibody also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.
- Antibodies also include single-armed composite monoclonal antibodies, single chain antibodies, including single chain Fv (sFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide, as well as diabodies, tribodies, and tetrabodies (Pack et al. (1995) J Mol Biol 246:28; Biotechnol 11: 1271;
- the antibodies are, e.g. , polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments, fragments produced by an Fab expression library, or the like.
- Aryl by itself or as part of another substituent, refers to a monovalent aromatic hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system, as defined herein.
- Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phen
- an aryl group comprises from 6 to 20 carbon atoms (C6-C20 aryl). In other embodiments, an aryl group comprises from 6 to 15 carbon atoms (C6-C15 aryl). In still other embodiments, an aryl group comprises from 6 to 15 carbon atoms (C 6 -Cio aryl).
- Arylalkyl by itself or as part of another substituent, refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with an aryl group as, as defined herein.
- Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-l-yl, 2-phenylethen-l-yl, naphthylmethyl, 2-naphthylethan-l-yl, 2-naphthylethen-l-yl, naphthobenzyl, 2-naphthophenylethan-l-yl and the like.
- an arylalkyl group is (C6-C30) arylalkyl, e.g. , the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C10) alkyl and the aryl moiety is (C6-C20) aryl.
- the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C10) alkyl and the aryl moiety is (C6-C20) aryl.
- an arylalkyl group is (C6-C20) arylalkyl, e.g. , the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (Ci-C 8 ) alkyl and the aryl moiety is (C6-C12) aryl.
- an arylalkyl group is (C6-C15) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C5) alkyl and the aryl moiety is (C6-C10) aryl.
- Aryldiyl by itself or as part of another substituent refers to a divalent hydrocarbon radical derived by the removal of one hydrogen atom from each of two different carbon atoms of a parent aromatic system or by removal of two hydrogen atoms from a single carbon atom of a parent aromatic ring system.
- the two monovalent radical centers or each valency of the divalent center can form bonds with the same or different atom(s).
- Typical aryldiyl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene,
- an aryldiyl group comprises from 5 to 20 carbon atoms. In other embodiments, an aryldiyl group comprises from 5 to 12 carbon atoms.
- Arylalkyldiyl refers to an acyclic alkyl diradical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with an aryl group.
- cLogD refers to a calculated value of LogD using the Chem Axon Physiochemical LogD/logP program. Note the two phases are not in equilibrium.
- cLogP refers to a computed partition coefficient of a compound between water and a lipophilic phase (usually octanol) using the Chem Axon Physiochemical LogD/LogP Program. Note the two phases are not in equilibrium.
- Chrographic matrix refers to conventional chromatography resins, such as, for example, reverse phase, silica, ion exchange or "mixed mode" supports. The chromatographic matrix may also include resins which include immobilized proteins (e.g. , serum albumin, alpha-acid
- glycoprotein or immobilized artificial membranes such as, for example, phospholipids (Pidgeon et al., Anal. Chem. 176 (1989)).
- ChrmLogD refers to a determination of LogD by chromatography.
- the chromLogD of an unknown compound is calculated by interpolation based of the retention time of two compounds of known eLogD that bracket the unknown compound and for which the retention time has been measured using the same HPLC method (ideally as a co-injection with the unknown compound).
- Chrom ss LogD refers to determination of a surrogate of eLogD of a compound by chromatography wherein the compound is covalently attached to a single stranded nucleic acid fragment, typically by an amide bond to a TEG linker. The retention time serves as the surrogate for eLogD of the compound when not attached to the single stranded nucleic acid.
- chrom ss LogD value of an unknown compound is calculated by interpolation based on the retention time of two compounds of known chromLogD that bracket the unknown compound and for which the retention time has been measured using the same HPLC method (ideally as a co-injection with the unknown compound) and an identical or functionally similar linker.
- the nucleic acid is 20 base pair DNA oligonucleotide and the surrogate of eLogD is chrom S s2oLogD.
- ChromdsLogD refers to determination of a surrogate of eLogD that is analogous to chrom ss LogD, with the key difference being that the conjugates are covalently attached to double stranded DNA fragment.
- the nucleic acid is 220 base pair DNA oligonucleotide, which is similar in design to a typical library gene used in Harbury et al. , United States Patent No. 7,479,472 and the surrogate of eLogD is chromd S 22oLogD.
- Coding template as used herein mean nucleic acid sequences which each comprise a plurality of hybridization sequences (i.e.
- hybridization sequences refer to oligonucleotides comprising between about 3 and up to 100, 3 and up to 50, and from about 5 to about 30 nucleic acid subunits.
- Such coding templates are capable of directing the synthesis of the combinatorial library based on the catenated hybridization sequences.
- the coding template is opera tively linked to a functional group or optionally a linking entity. Coding templates may be immobilized by capture templates and direct combinatorial library synthesis in DPCC.
- coding templates are oligonucleotides.
- the hybridization sequences are 20 nucleic acid subunits.
- hybridization sequences are separated by constant spacer sequences.
- Constant spacer sequences refer to oligonucleotides comprising between about 3 and up to 100, 3 and up to 50, and from about 5 to about 30 nucleic acid subunits.” refer to oligonucleotides comprising between about 3 and up to 100, 3 and up to 50, and from about 5 to about 30 nucleic acid subunits.
- the constant spacer sequences are 20 nucleic acid subunits.
- Combinatorial library refers to a library of molecules containing a large number, typically between 10 3 and 10 !5 or more different compounds typically characterized by different sequences of subunits, or a combination of different side chains functional groups and linkages. In some embodiments, a combinatorial library includes more than 10 2 molecules.
- Compounds refers to compounds encompassed by structural formulae disclosed herein and includes any specific compounds within these formulae whose structure is disclosed herein. Compounds may be identified either by their chemical structure and/or chemical name. When the chemical structure and chemical name conflict, the chemical structure is determinative of the identity of the compound.
- the compounds described herein may contain one or more chiral centers and/or double bonds and therefore, may exist as stereoisomers, such as double -bond isomers (i.e., geometric isomers), enantiomers or diastereomers.
- the chemical structures depicted herein encompass all possible enantiomers and stereoisomers of the illustrated compounds including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures.
- Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to the skilled artisan.
- the compounds may also exist in several tautomeric forms including the enol form, the keto form and mixtures thereof. Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated compounds.
- the compounds described also include isotopically labeled compounds where one or more atoms have an atomic mass different from the atomic mass conventionally found in nature. Examples of isotopes that may be incorporated into the compounds described herein include, but are not limited to, 2 H, 3 H, 13 C, 14 C, 15 N, 18 0, 17 0, 35 S, etc.
- eLogD refers to a determination of LogD by shake flask method in aqueous buffer/octanol mixture.
- Depsipeptide refers to a peptide as defined herein where one or more of amide bonds are replaced by ester bonds.
- Gel matrix as used herein includes, refers to various gels such as, cyrogels, agarose, superagarose or polyacrylamide gels.
- a gel matrix will include a lipid phase, such as, for example, vesicles, liposomes, micelles, lipophilic compounds, lipophilic polymers, artificial membranes or combinations thereof.
- Heteroalkyl Heteroalkanyl, Heteroalkenyl, Heteroalkanyl,
- Heteroalkyldiyl and Heteroalkyleno by themselves or as part of another substituent, refer to alkyl, alkanyl, alkenyl, alkynyl, alkyldiyl and alkyleno groups (and optionally any associated hydrogen atoms) are each, independently of one another, replaced with the same or different heteroatoms or heteroatomic groups.
- Typical heteroatoms or heteroatomic groups which can replace the carbon atoms include, but are not limited to, -0-, -S-, -N-, -Si-, -NH-, -S(0 , -S(0) 2 -,
- heteroatoms or heteroatomic groups may be placed at any interior position of the alkyl, alkenyl or alkynyl groups.
- cycloheteroalkyl substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl.
- Heteroaryl by itself or as part of another substituent, refers to a monovalent heteroaromatic radical derived by the removal of one hydrogen atom from a single atom of a parent heteroaromatic ring systems, as defined herein.
- Typical heteroaryl groups include, but are not limited to, groups derived from acridine, ⁇ -carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,
- the heteroaryl group comprises from 5 to 20 ring atoms (5-20 membered heteroaryl). In other embodiments, the heteroaryl group comprises from 5 to 10 ring atoms (5-10 membered heteroaryl).
- Exemplary heteroaryl groups include those derived from furan, thiophene, pyrrole, benzothiophene, benzofuran, benzimidazole, indole, pyridine, pyrazole, quinoline, imidazole, oxazole, isoxazole and pyrazine.
- Heteroarylalkyl by itself or as part of another substituent refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with a heteroaryl group. Where specific alkyl moieties are intended, the nomenclature heteroarylalkanyl, heteroarylakenyl and/or heteroarylalkynyl is used.
- the heteroarylalkyl group is a 6-21 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is (Ci-C 6 ) alkyl and the heteroaryl moiety is a 5-15-membered heteroaryl.
- the heteroarylalkyl is a 6-13 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety is (C1-C3) alkyl and the heteroaryl moiety is a 5-10 membered heteroaryl.
- Heteroaryldiyl refers to a divalent heteroaromatic group derived by the removal of one hydrogen atom from each of two different atoms of a parent heteroaromatic ring system or by the removal of two hydrogen atoms from a single atom of a parent heteroaromatic ring system.
- the two monovalent radical centers or each valency of the single divalent center can form bonds with the same or different atom(s).
- Typical heteroaryldiyl groups include, but are not limited to, divalent groups derived from acridine, arsindole, carbazole, ⁇ -carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, qui
- the heteroaryldiyl group is 5-20 membered heteroaryldiyl. In other embodiments the heteroaryldiyl group is 5-10 membered heteroaryldiyl. In some embodiments, heteroaryldiyl groups derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine are preferred.
- Heteroarylalkyldiyl refers to an acyclic alkyl diradical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with an heteroaryl group.
- Hydrates refers to incorporation of water into to the crystal lattice of a compound described herein, in stoichiometric proportions, resulting in the formation of an adduct.
- Methods of making hydrates include, but are not limited to, storage in an atmosphere containing water vapor, dosage forms that include water, or routine pharmaceutical processing steps such as, for example, crystallization (i.e. , from water or mixed aqueous solvents), lyophilization, wet granulation, aqueous film coating, or spray drying. Hydrates may also be formed, under certain circumstances, from crystalline solvates upon exposure to water vapor, or upon suspension of the anhydrous material in water. Hydrates may also crystallize in more than one form resulting in hydrate polymorphism. See, e.g. , (Guillory, K., Chapter 5, pp. 202-205 in Polymorphism in
- Hydrates may be characterized and/or analyzed by methods well known to those of skill in the art such as, for example, single crystal X-Ray diffraction, X-Ray powder diffraction, Polarizing optical microscopy, thermal microscopy, thermogravimetry, differential thermal analysis, differential scanning calorimetry, IR spectroscopy, Raman spectroscopy and NMR spectroscopy. (Brittain, H., Chapter 6, pp.
- Lipid matrix refers to lipophilic vesicles, artificial membranes, beads coated with lipophilic or amphophilic material, lipophilic compounds, lipophilic polymers, liposomes or micelles.
- LogD(pH) refers to a distribution of a compound between aqueous buffer and octanol at a given pH. Unless otherwise stated, the pH is assumed to be 7.4 and be written as LogD.
- Microx refers to, in general, at least three different types of matrixes: “chromatographic,” “gel,” or “lipid.”
- the matrix may be a hydrophilic liquid contacting a hydrophobic liquid (e.g. , octanol- water).
- Nucleic acid refers to an oligonucleotide analog as defined below as well as a double stranded DNA and RNA molecule.
- a DNA and RNA molecule may include the various analogs defined below.
- Oligos refer to nucleic acid oligomers containing between about 3 and up to about 300, and typically from about 5 to about 300 nucleic acid subunits.
- the oligos may include or be composed of naturally-occurring nucleotide residues, nucleotide analog residues, or other subunits capable of forming
- sequence-specific base pairing when assembled in a linear polymer, with the proviso that the polymer is capable of providing a suitable substrate for strand-directed polymerization in the presence of a polymerase and one or more nucleotide triphosphates, e.g. , conventional deoxyribonucleotides.
- known- sequence oligo is an oligo whose nucleic acid sequence is known.
- Oligonucleotide analog refers to a nucleic acid that has been modified and which is capable of some or all of the chemical or, biological activities of the oligonucleotide from which it was derived.
- An oligonucleotide analog will generally contain phosphodiester bonds, although in some cases, oligonucleotide analogs are included that may have alternate backbones.
- Modifications of the ribose-phosphate backbone may facilitate the addition of additional moieties such as labels, or may be done to increase the stability and half-life of such molecules.
- mixtures of naturally occurring nucleic acids and analogs can be made.
- mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
- the oligonucleotides may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence.
- the oligonucleotide may be DNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo-and ribo-nucleotides, and any combination of bases, including uracil, uridine, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.
- “Operatively linked,” as used herein, means at least two chemical structures joined together in such a way as to remain linked through the various manipulations described herein.
- a ligand or functional group and the coding nucleotide are linked covalently via an appropriate linker.
- the linker is at least a bivalent moiety with a site of attachment for the oligonucleotide and a site of attachment for the ligand or a functional group.
- the functional moiety is a polyarnide compound
- the polyamide compound can be attached to the linking group at the N-terminus, the C-terminus or via a functional group on one of the side chains.
- the linker is sufficient to separate the ligand and the oligon cleotide by at least one atom and in some embodiments by more than one atom. In most embodiments, the linker is sufficiently flexible to allow the ligand to bind target molecules in a manner which is independent of the oligonucleotide.
- Peptide refers to a polymer of amino acid residues between about 2 and 50 amino acid residues, between about 2 and 20 amino acid residues, or between about 2 and 10 residues.
- the amino acid residues may be any L- a- amino acid, D-a-amino residue, N-alkyl variants thereof or combinations thereof. In other embodiments, the amino acid residues may any L- a- amino acid, D-a-amino residue, ⁇ -amino acids, ⁇ - amino acids, N-alkyl variants thereof or combinations thereof.
- Parent aromatic ring system refers to an unsaturated cyclic or polycyclic ring system having a conjugated ⁇ electron system.
- parent aromatic ring system fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, fluorene, indane, indene, phenalene, etc.
- Typical parent aromatic ring systems include, but are not limited to, aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene and the like.
- Peptide nucleic acid refers to oligonucleotide analogues where the sugar phosphate backbone of nucleic acids has been replaced by psuedopeptide skeleton (e.g., N-(2-aminoethyl)-glycine)(Nielsen et al, U.S. Patent No. 5,539,082; Nielsen et al, U.S. Patent No. 5,773,571; Burchardt et al, U.S. Patent No. 6,395,474).
- psuedopeptide skeleton e.g., N-(2-aminoethyl)-glycine
- Protein refers to polymers of poly N-substituted glycine (Simon et al, Proc. Natl. Acad. Sci. (1992) 89(20) 9367-9371) and include cyclic variants thereof.
- Polypeptide refers to a polymer of amino acid residues typically comprising greater than 50 amino acid residues and includes cyclic variants thereof.
- Polypeptide includes proteins (including modified proteins such as glycoproteins, PEGylated proteins, lipoproteins, polypeptide conjugates with organic or inorganic ligands, etc.) receptor, receptor fragments, enzymes, structural proteins (e.g. , collagen) etc.
- the amino acid residues may be any L-a-amino acid, D-a-amino residue, or combinations thereof.
- the amino acid residues may be any L-a-amino acid, D-a-amino residue, N-alkyl variants thereof or combinations thereof.
- Recognition Element refers to an oligonucleotide, single or double- stranded RNA, single or double-stranded DNA, a DNA binding protein, a locked nucleic acid, a RNA binding protein, a peptide nucleic acid, a peptide, a depsipeptide, a polypeptide, an antibody, a peptoid, a polymer, a polysiloxanes, an inorganic compounds of molecular weight greater that 50 daltons, organic compounds of molecular weight between about 2000 daltons and about 50 daltons or a combination thereof.
- Salt refers to a salt of a compound, which possesses the desired pharmacological activity of the parent compound.
- Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethane-disulfonic acid,
- salts may be formed when an acidic proton present can react with inorganic bases (e.g. , sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, calcium hydroxide, etc.) and organic bases (e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N- methylglucamine, etc.).
- inorganic bases e.g. , sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, calcium hydroxide, etc.
- organic bases e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N- methylglucamine, etc.
- the salt is pharmaceutically acceptable.
- Solidvates refers to incorporation of solvents into to the crystal lattice of a compound described herein, in stoichiometric proportions, resulting in the formation of an adduct.
- Methods of making solvates include, but are not limited to, storage in an atmosphere containing a solvent, dosage forms that include the solvent, or routine pharmaceutical processing steps such as, for example, crystallization (i.e. , from solvent or mixed solvents) vapor diffusion, etc.
- Solvates may also be formed, under certain circumstances, from other crystalline solvates or hydrates upon exposure to the solvent or upon suspension material in solvent. Solvates may crystallize in more than one form resulting in solvate polymorphism. See, e.g., (Guillory, K., Chapter 5, pp. 205-208 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc., New York, NY, 1999)). The above methods for preparing solvates are well within the ambit of those of skill in the art, are completely conventional do not require any experimentation beyond what is typical in the art.
- Solvates may be characterized and/or analyzed by methods well known to those of skill in the art such as, for example, single crystal X-Ray diffraction, X-Ray powder diffraction, Polarizing optical microscopy, thermal microscopy, thermogravimetry, differential thermal analysis, differential scanning calorimetry, IR spectroscopy, Raman spectroscopy and NMR spectroscopy. (Brittain, H., Chapter 6, pp. 205-208 in Polymorphism in Pharmaceutical Solids, (Brittain, H. ed.), Marcel Dekker, Inc. New York, 1999).
- many commercial companies routinely offer services that include preparation and/or characterization of solvates such as, for example,
- Standard compound refers to, a compound of measured
- LogD which also has a measured retention time in a chromatographic matrix, or a measured R f in a gel matrix or a known absorbance in a lipid matrix both as a free compound and also when conjugated to a standard oligonucleotide (e.g. , an oligonucleotide of 20 nucleic acid subunits or 220 nucleic acid subunits) has also measured.
- Standard compounds are co-injected with combinatorial library members, for example, on a chromatographic matrix. Retention time of the library members can then be compared with retention time of standard compounds to provide an estimate of LogD of library members.
- Substituted when used to modify a specified group or radical, means that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent(s).
- substituent groups useful for substituting unsaturated carbon atoms in the specified group or radical include, but are not limited to, -R a , halo, -0-, -OR b , -SR b , -S-, -NR C R C , trihalomethyl, -CF 3 , -CN, -OCN, -SCN, -NO, -N0 2 , -N 3 , -S(0) 2 R b , -S(0) 2 0 " , -S(0) 2 OR b , -OS(0) 2 R b , -OS(0) 2 0 " , -OS(0) 2 OR b , -P(0)(0 ) 2 , -P(0)(OR b )(0 ), -P(0)(OR b )(OR b ), -C(0)R b , -C(S)R b , -C(NR b )R b , -C(0)0-
- Substituent groups useful for substituting nitrogen atoms in heteroalkyl and cycloheteroalkyl groups include, but are not limited to, -R a , -0 ⁇ , -OR b , -SR b , -S-, -NR C R C , trihalomethyl, -CF 3 , -CN, -NO, -NO2, -S(0) 2 R b , -S(0) 2 0-,
- Described herein are methods for estimating LogD of compounds which include a ligand operatively linked to a recognition element.
- the compounds may be members of combinatorial libraries and the methods may simultaneously provide estimates of LogD of a number of members of the combinatorial libraries.
- Combinatorial libraries are well known and may be synthesized by methods known in the art (Harbury, et al. , U.S. Patent No. 7,479,472; Liu et al,
- Patent No. 7,807,408 Liu et al, U.S. Patent No. 7,998,904; Liu et al, U.S.
- Patent No. 8,017,323 Liu et aL, U.S. Patent No. 8,183,178; Pedersen et al, U.S.
- the structure of the ligand may determine the relative LogD of members of combinatorial libraries.
- the recognition elements i.e., tags
- the recognition elements are typically isomeric polymers and thus possess similar physiochemical properties.
- ligands of different polarity may control the LogD of compounds, which include ligands operatively linked to recognition elements (Bouma et al, U.S. Patent No. 5,006,473, Pascoe et al, Electrophoresis 2003, 24, 4227-4240; Pascoe et al, Electrophoresis 2006, 27, 793-804).
- LogD of compounds which include a ligand operatively linked to a recognition element in combinatorial libraries may be estimated without modification or after binding of the recognition element to either a
- a method of estimating LogD of one compound which includes a ligand operatively linked to a recognition element is provided.
- the method includes contacting the compound with a lipid matrix, separating the compound absorbed by the lipid matrix from the compound not absorbed by the lipid matrix, measuring the amount of the compound absorbed by the lipid matrix and/or the amounts of the compound not absorbed by the lipid matrix where measurement of the amount of the compound absorbed by the lipid matrix and/or the amounts of the compound not absorbed by the lipid matrix provide an estimation of LogD of the compound.
- a method of estimating LogD of two or more compounds which include a ligand operatively linked to a recognition element includes contacting the compounds with a lipid matrix, separating compounds absorbed by the lipid matrix from compounds not absorbed by the lipid matrix, measuring the amounts of compounds absorbed by the lipid matrix and/or the amounts of compounds not absorbed by the lipid matrix where measurement of the amounts of compounds absorbed by the lipid matrix and/or the amounts of the compounds not absorbed by the lipid matrix provide an estimation of LogD of the compounds.
- an excipient is included in the contacting step.
- the excipient may be a cation or polycation such as, for example, a polyamine or a salt or a volume excluding agent such as PEG.
- the polyamine may be, but is not limited to, putrescine, cadaverine, spermidine or spermine.
- the salt is a fluoride, sulfate, phosphate, acetate, chloride, nitrate, bromide, chloride, perchlorate or thiosulfate anion combined with ammonium, potassium, sodium, lithium, magnesium, calcium or guanadinium cation.
- the salt is ammonium sulfate.
- the mixture is contacted with the matrix at a temperature between about 15 °C and about 50 °C.
- the matrix is a bead coated with lipid membrane material (e.g. , Longhi et al. , Drug Metabolism and Disposition 39:312-321, 2011), synthetic vesicles, liposomes or micelles.
- the synthetic vesicles are formed from mixtures of phosphatidylcholine, ceramide, phosphatidylethanolamine or phosphatidylserine.
- the synthetic vesicles are formed from mixtures of sodium dodecylsulfate, cetyltrimethylammonium bromide, sodium octylsulfate, octyl trimethyl ammonium bromide, l-palmitoyl-2-oleyl-sn-glycero-30-phosphocholine or bis (2-ethylhexyl) sodium sulfosuccinate.
- the synthetic vesicles are formed from mixtures of sodium dodecylsulfate and
- cetyltrimethylammonium sulfate In still other embodiments, micelles are formed from sodium dodecylsulfate and cetyltrimethylammonium bromide. In still other embodiments, synthetic vesicles are coated with carbohydrates, charged compounds or proteins. In still other embodiments, the matrix is an
- the lipid matrix must be reproducibly synthesized, be stable to the experimental conditions and have a dynamic range sufficient to resolve compounds of differing polarity. As such, the selection of a proper lipid matrix can involve routine experimentation, which is well within the ambit of the skilled artisan.
- Compounds absorbed by the matrix may be separated from compounds not absorbed by the matrix by methods including, but not limited to, centrifugation, filtration, electrophoresis or application of a magnetic field to magnetic beads.
- the amounts of compounds absorbed by the matrix and/or the amounts of compounds not absorbed by the matrix are measured by absorbance, fluorescence, radioactivity or quantitative mass spectrometry. In other embodiments, the amounts of compounds absorbed by the matrix and/or amounts of compounds not absorbed by the matrix are measured by DNA sequencing or qPCR.
- a method of estimating LogD of one compound which includes a ligand operatively linked to a recognition element comprises contacting the compound with a gel matrix, applying a voltage gradient to the gel matrix and measuring the R f of the compound on the gel matrix wherein the R f of the compound on the gel matrix provides an estimate of LogD of the compound.
- a method of estimating LogD of two or more compounds which include a ligand operatively linked to a recognition element includes the steps of contacting the compounds with a gel matrix, applying a voltage gradient to the gel matrix and measuring the R f of the compounds on the gel matrix wherein the R f of the compounds on the gel matrix provides an estimate of LogD of the compounds.
- the gel matrix includes a lipid phase.
- the lipid phase is synthetic vesicles, liposomes or micelles.
- compounds on the gel matrix are detected by staining.
- compounds on the gel matrix are detected by spectroscopic means (e.g. , fluorescence or absorbance).
- discrete regions of the gel matrix which include compounds are isolated.
- the compounds are eluted from each discrete isolated region of the gel matrix to provide a unique fraction, which is bar coded with a unique oligonucleotide, the fractions are pooled, amplified by the polymerase chain reaction and sequenced by NextGen sequencing.
- compounds are eluted from each discrete isolated region of the gel matrix to provide a unique fraction. The fractions are amplified by the polymerase chain reaction and sequenced by NextGen sequencing
- a method of estimating LogD of one compound which include a ligand operatively linked to a recognition element includes the steps of contacting the compound with a
- a method of estimating LogD of two or more compounds which include a ligand operatively linked to a recognition element includes the steps of contacting the compounds with a chromatographic matrix and measuring the retention times of the compounds on the chromatographic matrix wherein the measured retention times of the compounds on the chromatographic matrix provides an estimate of LogD of the compounds.
- a proper chromatographic matrix is within the ambit of the skilled artisan and is often a matter of trial and error. Elution with discrimination of recognition elements operatively linked to ligands is an essential requirement of a chromatographic matrix. Another feature of importance is pore size. In some embodiments, the pore size of the chromatographic matrix is about 100 A. In other embodiments, the pore size of the chromatographic matrix is about 4000 A. In some embodiments, the recognition element is a single stranded DNA oligonucleotide, the ligand is a peptide or organic molecule and the
- chromatographic matrix is reverse phase chromatographic support.
- the retention time of the compounds is chrom SS xxLogD wherein xx is the number of nucleotides in the DNA oligonucleotide. In some embodiments, xx is 20.
- two or more standard compounds are included in the contacting step where the standard compounds include ligands of measured chrom SS xxLogD and measured chromLogD.
- the chromatographic matrix is packed into a high pressure liquid chromatography column. In still other embodiments, fractions including the compounds are collected by elution of the column.
- each fraction is amplified by the polymerase chain reaction and sequenced by NextGen sequencing to provide an estimate of the retention time of the compounds in the fraction.
- the retention times of the compounds are compared with the retention times of the standard compounds to provide an estimate of chrom SS xxLogD of the compounds.
- a unique oligonucleotide bar code is attached to compounds in each fraction, the fractions are pooled, amplified by the polymerase chain reaction and sequenced by NextGen sequencing to provide an estimate of the retention time of the compounds in the fractions.
- the retention times of the compounds are compared with the retention times of the standard compounds to provide an estimate of chrom SS xxLogD of the compounds.
- the recognition element is double stranded DNA
- the ligand is a peptide or organic molecule
- the chromatographic matrix is a reversed phase chromatographic support.
- the retention time of the compounds is chromdsxxLogD.
- xx is 220.
- two or more standard compounds are included in the contacting step where the standard compounds include ligands of measured chromdsxxLogD and measured chromLogD.
- the chromatographic matrix is packed into a high pressure liquid chromatography column.
- fractions including the compounds are collected by elution of the column.
- each fraction is amplified by the polymerase chain reaction and sequenced by NextGen sequencing to provide an estimate of the retention time of the compounds in the fraction.
- the retention times of the compounds are compared with the retention times of the standard compounds to provide an estimate of chromdsxxLogD of the compounds.
- a unique oligonucleotide bar code is attached to compounds in each fraction, the fractions are pooled, amplified by the polymerase chain reaction and sequenced by NextGen sequencing to provide an estimate of the retention time of the compounds in the fractions.
- the retention times of the compounds are compared with the retention times of the standard compounds to provide an estimate of chromd SXX LogD of the compounds.
- the lipophilicity or LogD of a compound which includes a ligand operatively linked to a recognition element may be estimated in a number of different configurations, including, but not limited, to the following.
- the lipophilicity or LogD of a compound which includes a ligand operatively linked to a recognition element may be estimated without any modification.
- the lipophilicity or LogD of a compound which includes a ligand operatively linked to a recognition element may be estimated after binding to a complementary moiety.
- the lipophilicity or LogD of a compound which includes a ligand operatively linked to a recognition element may be estimated after binding to a modified complementary moiety, such as, for example, a complementary moiety with an attached group, which in some embodiments may be hydrophobic.
- the recognition element is an oligonucleotide, single stranded DNA or single stranded RNA, which is operatively linked to a first ligand at the 3' terminus.
- oligonucleotide, single stranded DNA or single stranded RNA is operatively linked to the first ligand at the 3' terminus is hybridized with a complementary oligonucleotide or single stranded DNA operatively linked to a second ligand at the adjacent 5' terminus prior to contacting the mixture of compounds with the lipid matrix.
- the recognition element is an oligonucleotide, single stranded DNA or single stranded RNA, which is operatively linked to a first ligand at the 5' terminus.
- the oligonucleotide, single stranded DNA or single stranded RNA is operatively linked to the first ligand at the 5' terminus is hybridized with a complementary oligonucleotide or single stranded DNA operatively linked to a second ligand at the adjacent 3' terminus prior to contacting the mixture of compounds with the matrix.
- the first ligand can be at the 3' terminus and the second ligand may be at the 3' terminus.
- the second ligand may be, but is not necessarily identical, for all members of the library.
- the second ligands of the combinatorial library, supra have similar octanol- water coefficients (log D).
- the second ligands may have octanol-water coefficients (log D) greater than 1, greater than 2, greater than 3, greater than 4, greater than 5 or greater than 6.
- the second ligands of the combinatorial library, supra are identical. In other of these embodiments, the second ligands are hydrophobic.
- the second ligands have octanol-water coefficients (log D) greater than 5 or greater than 6.
- the second ligands are C6-C30 alkanyl, Ci 8 alkanyl, aryl, arylalkyl, sterol derivatives, steroid derivatives or cholesterol derivatives.
- a hydrophobic 3' constant second ligand may confer measurable distribution of double stranded DNA into a membrane fraction which may in the middle of the dynamic range of a membrane based assay.
- the proximity of the 5' variable first ligand may perturb the affinity of the 3' constant second ligand for the membrane fraction thus, in principle, allowing for measurement of the lipophilicity of the 5' variable first ligand and in some embodiments, LogD of the 5' variable first ligand.
- a double stranded DNA molecule comprising one oligonucleotide operatively linked to a variable first ligand at the 3' terminus hybridized to a complementary oligonucleotide operatively linked to a constant second ligand at the adjacent 5' terminus where the LogD of the second ligand is greater than 6 is provided.
- above molecule is used in a method of estimating LogD of two or more first ligands of double stranded DNA molecules.
- the method includes the steps of contacting the double stranded DNA molecules with a lipid matrix, separating the double stranded DNA molecules absorbed by the lipid matrix from double stranded DNA molecules not absorbed by the lipid matrix and measuring the amounts of double stranded DNA molecules absorbed by the lipid matrix and/or the amounts of double stranded DNA molecules not absorbed by the matrix wherein measurement of the amounts of double stranded DNA molecules absorbed by the matrix and/or the amounts of the double stranded DNA molecules not absorbed by the matrix provide an estimate of LogD of the first ligands of the double stranded DNA molecules.
- use of at least two standard compounds, which in some embodiments are operatively linked to recognition elements may provide estimates of LogD of unknown compounds, which include a ligand operatively linked to a recognition element, by interpolation between the LogD values of the standards.
- use of a standard compound with a LogD value of 3 and another standard compound with a LogD value of 4 would allow for identification of compounds of LogD between 3 and 4.
- standard compounds of known LogD when attached to recognition elements known in the art and known R f , may be used to bracket the R f , of compounds on a gel matrix and hence to estimate the LogD of the compounds.
- standard compounds of known LogD may be used to bracket the retention time of the compounds on the chromatographic matrix and hence to estimate LogD of the compounds.
- compounds, which include a ligand operatively linked to a recognition element may be affinity purified by binding to a target, which may be a biological target, such, as for example, a receptor, an enzyme, a protein, a cell, a membrane preparation, etc. , prior to contacting with a matrix.
- affinity purification enriches the mixture of compounds by removing non-binding members, hence providing lipophilicity estimates only for compounds which have demonstrated affinity for the target.
- affinity purification enriches the mixture of compounds by removing non-binding members, hence providing lipophilicity estimates only for compounds which have demonstrated affinity for the target.
- the above methods may be used in either a selection mode or a screening mode.
- a library of compounds may be applied to a gel matrix including a lipid phase, separated by electrophoresis, desired regions of the gel isolated and the nucleic tags on the corresponding compounds PCR amplified. Sequencing and subsequent correlation of sequence with mobility on the gel matrix enables assignment of LogD to members of the compound library in the selected region of the gel.
- a library of compounds may be contacted with a lipid matrix, the population that is bound to the lipid matrix separated, PCR amplified and used as input for another round of translation and selection on a lipid matrix.
- a library of compounds may be contacted with a chromatographic matrix, fractions collected, PCR amplified and used as input for another round of translation and selection on a chromatographic matrix. Iteration of such methods may exponentially enrich for compounds with a desired LogD.
- the PCR amplified material may be used as input, for example, affinity selection.
- the above methods can also be used to measure the LogD of a single compound which includes a ligand operatively linked to a recognition element as well as compounds that are members of a combinatorial library (i.e. , a complex mixture).
- a plurality of compounds, each linked to a unique oligonucleotide are synthesized and pooled.
- the pooled compounds may now be separated on the basis of differential LogD using any of the methods described above. Sequencing of the collected fractions provides the relative abundance of any given oligonucleotide in any given fraction, which then enables a calculation to estimate LogD. Because each compound is attached to a unique
- oligonucleotide a plurality of compounds can be assayed in parallel.
- the pooled compounds may affinity purified, for example, against an immobilized target, prior to separation by the methods described herein.
- a mixture of compounds is prepared by synthesizing compounds attached to an invariant oligonucleotide in separate vessels by methods know in the art, supra.
- the invariant oligonucleotide may be primed with unique oligonucleotides that include a unique bar code region interposed between flanking oligonucleotide regions identical to the above invariant oligonucleotide and converted to double stranded material by conventional methods.
- the double stranded material is then pooled and used in any of the above methods to provide estimates of LogD for the compounds.
- the double stranded material may be affinity purified against a biological target, before being used in any of the methods described above.
- any of the above methods may be used to provide relative lipophilicities of combinatorial library compounds without estimation of LogD when standard compounds are not used or are unavailable.
- standard compounds are included in the contacting steps.
- the ligand is an oligonucleotide, single stranded RNA, single stranded DNA, double stranded RNA, double stranded DNA, a peptide, a depsipeptide, a peptoid or an organic compound of molecular weight of less than 2000 daltons. In other embodiments, the ligand is a peptide, a peptoid or an organic compound of molecular weight of less than 2000 daltons. In still other embodiments, the ligand is a peptide or an organic compound of molecular weight of less than 2000 daltons.
- the ligand is operatively linked to a recognition element with a linker, which generally is any molecule or substance which performs the function of connecting the ligand to the recognition element.
- the distance between the ligand and the recognition element may be greater than about 10A, about 25 A, about 50 A or about 100 A.
- the linker may vary in structure and length.
- the linker may be hydrophobic or hydrophilic, long or short, rigid, semi rigid or flexible, etc.
- the linking group can comprise, for example, a polymethylene chain, such as a— (CH 2 ) n — chain or a poly(ethylene glycol) chain, such as chain, where in both cases n is an integer from 1 to about 20, 5'-0-Dimethoxytrityl- l ⁇ 2'-Dideoxyribose-3'-[(2-cyanoethyl)-(N,N-diisopropyl)J-phosphoramidite; 9- O-Dimethoxytrityl-triethylene glycol, l-[(2-cyanoethyl)-(N,N-isopropyl)]- phosphoramidite; 3-(4,4'-Dimethoxytrityloxy)propyl- l-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite; and ⁇ 8-O-Dimethoxytritylhexaethyleneglycol, 1 ,- [(2-
- the recognition element in broadest terms, may be an oligonucleotide, a double stranded oligonucleotide, single stranded RNA, single stranded DNA, double stranded DNA, a double stranded RNA-DNA hybrid, a DNA binding protein, a RNA binding protein, a peptide nucleic acid, a peptide, a depsipeptide, a polypeptide, locked nucleic acids, an antibody or a peptoid.
- the recognition element is an oligonucleotide, a double stranded oligonucleotide, single stranded RNA, a double stranded RNA-DNA hybrid, single stranded DNA, double stranded DNA or a peptide nucleic acid. In other embodiments, the recognition element is an oligonucleotide, single stranded DNA, single stranded RNA or double stranded DNA.
- the compounds described above may be members of combinatorial libraries and the method may simultaneously provide estimates of the lipophilicity of more than one member(s) of those particular combinatorial libraries.
- Combinatorial libraries include, but are not limited to, tagged combinatorial libraries described, supra.
- the recognition element may include, but is not limited to, all tags or labels previously described in the art and all methods used to prepare such tags.
- the precise chemical structure of the recognition element will not be of determinative importance in the methods described herein. Accordingly, the methods described herein may be used with any tagged combinatorial library, including those not yet known in the art.
- identification of the compound(s) operatively linked to recognition element(s) can be accomplished by
- recognition elements may be identified by any method know to those of skill in the art including, for example, but not limited to, biological methods (e.g., affinity binding, sequencing, etc.) and chemical methods (e.g. , NMR, mass spectroscopy, etc.).
- identification involves amplification and sequencing amplified recognition elements or quantitative hybridization to complementary sequences. Amplification of nucleic acids, sequencing of nucleic acids and quantitative hybridization are conventional and are well known in the art.
- RE is a recognition element
- L a is a ligand
- Ci and C2 are independently connecting elements
- n and o are independently 0 or 1 ;
- Xi, X2 and X3 are functional groups.
- the compound of Formula (i) is a coding template used to direct synthesis of small-molecule combinatorial libraries (Harbury, et al. , U.S. Patent No. 7,479,472).
- Coding templates are compounds having a nucleic acid sequence containing at least one, typically two or more different catenated hybridization sequences, optional constant spacer sequences and an attached linking entity or functional group (i.e. , chemical reaction moiety) (FIG. 1 ).
- Coding templates are not limited in the number of hybridization sequences and/or constant spacer sequences.
- the hybridization sequences in any given coding template generally differ from the sequences in any other coding template. It should be noted that different coding templates can share a common codon.
- the hybridization sequences of each coding template identify the particular chemical compounds used in each successive synthesis step for synthesizing a unique ligand attached to the linking entity or functional group. As such, hybridization sequences of each coding template also identify the order of attachment of the particular chemical units to the linking entity or functional group.
- the compound of Formula (I) is a tagging oligonucleotide, such as those used in methods described in Morgan et al , U.S. Patent No. 7,972,992; Morgan et al , U.S. Patent No. 7,935,658; Morgan et al , U.S. Patent Application No. 2011/0136697; Morgan et al, U.S. Patent No.
- the compound of Formula (I) is an
- Xi is attached to a functional group on RE.
- X2 or X3 are attached to a functional group on L a .
- the functional group on RE is OH, NH 2 or NR X where X is an alkyl group or a methyl group.
- RE and L a are as defined, supra.
- X 1 is -C(O)-, -CONR1-, -C(0)0-, -P(0)NR 2 -, -P(0)(OH)NR 3 , -P(0)0-, -P(0)(OH)0-, -S(0) 2 NR4-, -OC(S)-, -OC(0)0-, -OC(S)0-,-OC(0)NR 5 -, -NR 6 C(0)0-, -NR 7 C(0)NR 8 -, -OP(0)NR 9 -,
- X 2 is -C(0 , -CONR 2 i-, -C(0)0-, -P(0)NR 22 -, -P(0)(OH)NR 23 , - ⁇ (0)0-, -P(0)(OH)0-, -S(0) 2 NR 24 -, -OC(S)-, -OC(0)0-, -OC(S)0-,-OC(0)NR 25 -, -NR 26 C(0)0-, -NR 27 C(0)NR 28 -, -OP(0)NR 29 -, -OP(O)(OH)NR 30 -, -OP(0)0-, -OP(0)(OH)0-, -NR 3 i-,
- X 3 is -C(0 , -CONR41-, -C(0)0-, -P(0)NR 42 -, -P(0)(OH)NR 4 , - ⁇ (0)0-, -P(0)(OH)0-, -S(0) 2 NR 44 -, -OC(S)-, -OC(0)0-, -OC(S)0-,-OC(0)NR 45 -, -NR 46 C(0)0-, -NR 47 C(0)NR 48 -, -OP(0)NR 49 -, -OP(O)(OH)NR 50 -, - ⁇ (0)0-, -OP(0)(OH)0-, -NR51-, -NR 52 P(0)0-, -NR 53 P(0)(OH)0-, -S-, -O- or a carbon-carbon bond; and Ri-Ri 3 , R 2 i-R 33 and R 4 i
- Ci and C 2 are independently alkyldiyl, substituted alkyldiyl, aryldiyl, substituted aryldiyl, arylalkyldiyl, substituted arylalkydiyl, heteroalkyldiyl, substituted heteroalkyldiyl, heteroarylalkydiyl or substituted heteroarylalkyldiyl.
- RE is an oligonucleotide, single or double- stranded RNA or single or double- stranded DNA
- L a is a peptide, peptoid or an organic compound of molecular weight of less than 2000 daltons
- Ci and C2 are independently alkyldiyl, substituted alkyldiyl, aryldiyl, substituted aryldiyl, arylalkyldiyl, substituted arylalkydiyl, heteroalkyldiyl, substituted
- Xi is -C(0 , -CONR1-, -C(0)0-, -P(0)(OH)NR 3 - or -P(0)(OH)0-;
- X 2 is -C(O)-, -CONR21-, -C(0)0-, -P(0)(OH)NR 23 - or -P(0)(OH)0-;
- X 3 is -C(O)-, -CONR41-, -C(0)0-, -P(0)(OH)NR 43 - or -P(0)(OH)0-.
- RE is an oligonucleotide
- X 1 is -P(0)(OH)0-
- Ci is -(CH 2 )20(CH2)20(CH 2 )3
- X2 is -NHC(O)-
- o is 0
- L a is cholesterol.
- RE is an oligonucleotide
- X 1 is -P(0)(OH)0-
- Ci is -(CH 2 )20(CH2)20(CH 2 )3
- X2 is - NHP(0)(OH)0-
- L a is -nCi 8 H 3 7.
- RE is an oligonucleotide
- Xi is -P(0)(OH)0-
- Ci is -((CH 2 )20)2(CH 2 )3-
- X2 is -NHC(O)-
- C 2 is n-C 3 H 6 , n-C 7 Hi 4 or n-CnH 22
- X 3 is -NHC(O)-.
- L a is a peptide, peptoid or an organic compound of molecular weight of less than 2000 daltons.
- L a is a sterol derivative or a cholesterol derivative.
- L a is a compound derived from
- RE is an oligonucleotide
- Xi is -P(0)(OH)0-
- Ci is
- L a is a peptide, peptoid or an organic compound of molecular weight of less than 2000 daltons.
- L a is a sterol derivative or a cholesterol derivative.
- L a is a compound derived from
- RE is an oligonucleotide
- Xi is -P(0)(OH)0-
- Ci is -((CH 2 )20)2(CH 2 )3-
- X 2 is -NHC(O)-
- C 2 is piperdinyl or -(CH 2 ) 3 -piperdinyl
- X3 is -C(O)-.
- L a is a peptide, peptoid or an organic compound of molecular weight of less than 2000 daltons L a .
- L a is a compound derived from
- RE is an oligonucleotide
- Xi is -P(0)(OH)0-
- Ci is -C6H12-, -C12H24- or C18H36-
- X2 is -NH-.
- RE is an oligonucleotide
- Xi is -P(0)(OH)0-
- Ci is -((CH2)20) 5 CH 2 CH2-
- X 2 is - P(0)(OH)0-
- L2 is C12H24
- X3 is -NH-.
- L a is a peptide, peptoid or an organic compound of molecular weight of less than 2000 daltons.
- a library of compounds including more than one compound of Formula (I) where RE and L a are different is provided.
- Ci, C2, n, o, Xi, X2 and X3 are identical in each compound of the library.
- REc is a recognition element
- Lb is a ligand with similar or identical hydrophobicity
- C3 and C 4 are independently linkers
- X 4 , X5 and X 6 are functional groups
- X 4 is attached to a functional group on RE.
- X5 or X 6 are attached to a functional group on Lb.
- the functional group on RE C is OH, N3 ⁇ 4 or NR y where Y is an alkyl group or a methyl group.
- RE and Lb are as defined, supra.
- Lb is a peptide or an organic compound of molecular weight of less than 2000 daltons. In other embodiments, Lb is a hydrophobic compound with a log P greater than 4, greater than 5 or greater than 6.
- X 4 is -C(O)-, -CONRei-, -C(0)0-, -P(0)NR 62 -,
- X 5 is -C(O)-, -CONRsi-, -C(0)0-, -P(0)NR 82 -, -P(0)(OH)NR 83 , -P(0)0-, -P(0)(OH)0-, -S(0) 2 NR 8 4-, -OC(S)-, -OC(0)0-, -OC(S)0-,-OC(0)NR 85 -, -NR 86 C(0)0-, -NR 87 C(0)NR 88 -, -OP(0)NR 89 -, -OP(O)(OH)NR 90 -, -OP(0)0-, -OP(0)(OH)0-, -NR91-,
- X 5 is -C(O)-, -CONR91-, -C(0)0-, -P(0)NR 92 -, -P(0)(OH)NR 93 , - ⁇ (0)0-, -P(0)(OH)0-, -S(0) 2 NR 94 -, -OC(S)-, -OC(0)0-, -OC(S)0-,-OC(0)NR 95 -, -NR 96 C(0)0-, -NR 97 C(0)NR 98 -, -OP(0)NR 99 -, -OP(0)(OH)NRioo-, - ⁇ (0)0-, -OP(0)(OH)0-, -NR101-, -NRio 2 P(0)0-, -NRio3P(0)(OH)0-, -S-, -O- or a carbon-carbon bond and R 7 i-R 7 3,
- Ci and C 2 are independently alkyldiyl, substituted alkyldiyl, aryldiyl, substituted aryldiyl, arylalkyldiyl, substituted arylalkydiyl,
- heteroalkyldiyl substituted heteroalkyldiyl, heteroarylalkydiyl or substituted heteroarylalkyldiyl.
- RE C is an oligonucleotide, single stranded RNA or single stranded DNA
- Lb is a peptide or an organic compound of molecular weight of less than 2000 daltons
- Ci and C 2 are independently alkyldiyl, substituted alkyldiyl, aryldiyl, substituted aryldiyl, arylalkyldiyl, substituted arylalkydiyl, heteroalkyldiyl, substituted heteroalkyldiyl, heteroarylalkydiyl or substituted heteroarylalkyldiyl
- X 4 is -C(O)-, -CONR1-, -C(0)0-, -P(0)(OH)NR 3 - or -P(0)(OH)0-
- X 5 is -C(O)-, -CONR 2 i-, -C(0)0-, -P(0)(OH)NR 23 - or
- -P(0)(OH)0- and X 6 is -C(O)-, -CONR41-, -C(0)0-, -P(0)(OH)NR 43 - or
- Lb is C6-C30 alkanyl, Ci 8 alkanyl, aryl, arylalkyl, a sterol derivative, a steroid derivative or a cholesterol derivative.
- a library of compounds including more than one compound of Formula (II) where RE C is different and Lb is of similar hydrophobicity is provided.
- C3, C 4 , k, 1, X 4 , X5 and X 6 are identical in each compound of the library.
- a compound comprising a compound of Formula (I) hybridized to a compound of Formula (II) wherein RE and RE C are independently an oligonucleotide, single stranded RNA or single stranded DNA, Ci, C 2 , n, o, Xi, X 2 , X3, L a , Lb C3, C 4 , k, 1, X 4 , X5 and X 6 are as defined above is provided.
- a library of compounds including more than one compound of Formula (I) hybridized to a compound of Formula (II) is provided.
- compounds where RE, RE C and L a are different and Lb is of similar hydrophobicity is provided.
- Lb is of similar
- Ci hydrophobicity
- Ci C 2 , n, o, Xi, X 2 , X3, C3, C 4 , k, 1, X 4 , X5 and X 6 are identical in each compound of the library.
- Lb, Ci, C 2 , n, o, Xi, X 2 , X3, C3, C 4 , k, 1, X 4 , X5 and X 6 are identical in each compound of the library.
- the recognition elements are DNA oligonucleotides with connectivity to the ligand or linkers to the 5 'end of the oligomers.
- X is 5'GCTCGTCGCATTCGGCACGC-3' (SEQ. ID. No. 1), Y is
- Z is ATGGTATCAAGCTTGCCACAGCCGAAGCAGACTTAATCACGTCGAGC TCTCTCTACTGCATAGATTAGCGTACATAGGCCCGGAACCCGGGACAAG GTGTCATCATAGATGTCAGCACTGGGTAGTGGCCTGCAGCTATGTAAA TCACGCTTGGTAAGTTGGGTAATTCTGTACAGGTCGCGATAATCAGCG GGAATCAGGCGGCAGAATCTCGAGTACTAG (SEQ ID. No. 4).
- the resin was washed with water, lOOmM N- methyl morpholine in water, water, 50% water/MeOH, MeOH, 70% MeOH/DMF, 50% MeOH/DMF and spun dry in a centrifuge. In the meantime, a suspension of 12.1 mg Fmoc-12- aminododecanoic acid (Fmoc-12-Ado-OH) in 139uL DMF (not fully soluble) was prepared. 278 uL N-methyl morpholine solution (10 uL in 909 uL methanol) was added but the solution was still not soluble. The mixture was sonicated but still did not dissolve.
- the suspension (187.5 uL) was aliquoted and treated with 62.5 uL freshly prepared DMTMM (4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4- methylmorpholinium) solution (5.2 mg in 157 ul DMF). Then 40 uL aliquots of the resulting suspension were added to each well, which were then kept at room temperature for 1 hour.
- DMTMM 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4- methylmorpholinium
- the resin was filtered, rinsed with 50% MeOH/DMF, 70% MeOH/DMF, MeOH, 50% MeOH/water, water (allowing to stand 5 minutes), 50%
- the resin was washed with water, 100 mM N-methyl morpholine in water, water, 50% water/MeOH, MeOH, 70% MeOH/DMF, 50% MeOH/DMF and then spun dry in a centrifuge. In the meantime, a solution of 11.2 mg Fmoc- 12-amino-4,7,10-trioxadodecanoic acid in 127 uL DMF was prepared. 62.5 uL of this solution was aliquoted and treated with 125 uL N-methyl morpholine solution (10 uL N-methyl morpholine in 909 uL methanol) followed by 62.5 uL freshly prepared DMTMM solution (5.2 mg in 157 ul DMF). Aliquots (40 uL) of the resulting solution were added to each well, which were then kept at room temperature for 1 hour.
- the resin was filtered, rinsed with 50% MeOH/DMF, 70% MeOH/DMF, MeOH, 50% MeOH/water, water (allowed to stand for 5 minutes), 50%
- MeOH/water MeOH, 70% MeOH/DMF, 50% MeOH/DMF and spun down.
- the oligomer was eluted from each well with 3 x 33 uL of l.5M NaCl/10 mM ammonium acetate in water, allowed to stand for 2 minutes after each solvent addition prior to filtration.
- the crude oligomer was purified by RP- HPLC on a Phenomenex Luna C-18 column (Solvent A: lOmM ammonium acetate in water; Solvent B: acetonitrile).
- the desired fractions were dried via Speedvac.
- the resultant compound was analyzed by HPLC essentially as described in Figure 8 and the purity was judged to be >90% as quantified by the area percent corresponding to the major peak.
- the measured mass of the compound in the major peak was within experimental error of that predicted for Fmoc-12-amino-4,7,10-trioxadodecanoic acid modified oligomers.
- the resin was washed with water, 100 mM N-methyl morpholine in water, water, 50% water/MeOH, MeOH, 70% MeOH/DMF, 50% MeOH/DMF and then spun dry in a centrifuge. In the meantime, a solution of 12.6 mg 4-(l- Fmoc-piperidin-4-yl)butanoic acid in 159 uL DMF was prepared. 62.5 uL of this solution was aliquoted and treated with 125 uL N-methyl morpholine solution (10 uL N-methyl morpholine in 909 uL methanol) and then 62.5 uL freshly prepared DMTMM solution (4.5 mg in 136 ul DMF). Then, 40uL aliquots of the resulting solution were added to each well, which were then kept at room temperature for 1 hour. The resin was filtered, rinsed with 50% MeOH/DMF, 70% MeOH/DMF,
- MeOH/water MeOH, 70% MeOH/DMF, 50% MeOH/DMF and spun down.
- the oligomer was eluted from each well with 3 x 33 uL of l.5 M NaCl/10 mM ammonium acetate in water, allowed to stand for 2 minutes after each solvent addition prior to filtration.
- the crude oligomer was purified by RP- HPLC on a Phenomenex Luna C-18 column (Solvent A: 10 mM ammonium acetate in water; Solvent B: acetonitrile). The desired fractions were dried via Speedvac.
- EXAMPLE 4 General Procedure for Coupling Acetic or Bile Acids to Extended Oligomers
- the resin was washed with water, 50% MeOH/water, MeOH, 70% MeOH/DMF, 50% MeOH/DMF, 30% MeOH/DMF and DMF and spun dry in a centrifuge. 60 uL 20% piperidine in DMF was added to the well, and allowed to stand at room temperature for 10 minutes.
- the resin was filtered, then washed with DMF, 30% MEOH/DMF, 50% MeOH/DMF, 70% MeOH/DMF, MeOH, 50% MeOH/water, water, 50%
- the resin was filtered, rinsed with 50% MeOH/DMF, 70% MeOH/DMF, MeOH, 50% MeOH/water, water (allowed to stand 5 minutes), 50%
- MeOH/water MeOH, 70% MeOH/DMF, 50% MeOH/DMF and spun down.
- the oligomers were eluted from the well with 3 x 33uL 1.5 M NaCl/10 mM ammonium acetate in water and allowed to stand for 2 minutes after each solvent addition prior to filtration. The resulting oligomers were analyzed by HPLC and Nanodrop (for concentration).
- Analytical data from an exemplary conjugate is given in Figure 8.
- a conjugate of cholic acid with ZappT was prepared and purified essentially as described in Example 4. The trace shows the elution profile of a cholic acid conjugate. Time in minutes is given on the x-axis. Percent Solvent B is given on the y-axis (right side; axis runs from zero to 100 percent). Solvent A was 10 mM ammonium acetate in water. Solvent B was acetonitrile.
- lx TBE Trimeda-borate-EDTA electrophoresis buffer, Maniatis et al. , p. 454
- the solution is cooled overnight to room temperature and then filtered through a 0.2 micron filter.
- Vesicles prepared in this manner are reported to be stable for multiple days at room temperature.
- Low melting agarose (0.75 gm) is dissolved in 25 mL lx TBE and placed in a water bath at 50° C along with a separate tube of vesicles. After equilibrium is reached, the tubes are mixed in a preheated flask, gently swirled and poured into a standard horizontal mini-gel apparatus to provide a vesicle agarose gel ((standard lx TBE, 1.5% agarose, 0.9%). After the gels have solidified, samples are loaded, run at 100 volts, stained with ethidium bromide and photographed by standard methods (Molecular Cloning; Maniatis et al. ; Cold Spring Harbor press, 1982).
- the gels indicate that appendage of either cholesterol (Choi) or Ci 8 (n-CisEbv) to the 5' end of a DNA molecule (i.e. , compounds 1 and 2, respectively) can greatly retard the mobility of the conjugates in agarose gels containing 0.9% CTAB/SOS vesicles, relative to reference gels lacking these vesicles.
- FAM is 5'- 6-FAM (Fluorescein).
- the 6- FAM moiety is comprised of a single isomer derivative of fluorescein and is the most commonly used fluorescent dye attachment for oligonucleotides and is compatible with most fluorescence detection equipment. It becomes protonated and has decreased fluorescence below pH 7: it is typically used in the pH range 7.5-8.5.
- FAM can be attached to 5' or 3 ! end of oligos
- the partitioning coefficient into the embedded vesicles may be obtained, which may act as a surrogate for diffusion into other lipid membranes. Accordingly, one may in principle, use this method to separate a plurality of DNA molecules with different ligands on gel systems of this type. Sequencing of isolated gel sections can then enable the inference of the mobility of DNA-conjugate species included therein.
- EXAMPLE 8 Matrix Method for Estimation of Lipophilicity of Steroids Operatively linked to a Recognition Element
- EXAMPLE 10 General Procedure for Conversion of Oligonucleotides of 20 Nucleic Acid Subunits Operatively Linked to a Ligand to Oligonucleotides of 220 Nucleic Acid Subunits Operatively Linked to a Ligand
- HPLC purified 20 base pair single stranded oligomers operatively linked to alkyl or steroid ligands were transformed into a double stranded 220 base pair gene via polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- the oligomers operatively linked to alkyl or steroid ligands and a 20 base pair reverse oligomer (luM) were used as PCR primers in a lOOuL reaction with 10 ng of double stranded 220 base pair gene template with 2x Fusion Flash High-Fidelity PCR Master Mix
- ThermoFisher F548S Thermocycling was performed at 98 C° for 10s, followed by 10 cycles of 98 C°, Is, 55 C°, 5s, 72 C°, 10s then followed by an additional protocol of 98 C° for 10s followed by 15 cycles of 98 C°, Is, 50 C°, 5s, 72 C°, 10s.
- PCR products of 220 base pair were confirmed via gel electrophoresis, purified with the QIAquick PCR Purification Kit (Qiagen) and DNA concentration was quantified via nanodrop A260 measurements.
- Chrom2oLogD may be determined by extensions of the above experiment.
- a set of 9 linear peptides (N terminal acetylated and C terminal amide) were synthesized using standard methods and cLogD and eLogD values determined using conventional procedures.
- the eLogD of FFFF could not be determined because of formation of a visible aggregate at the octanol- water interface.
- Extended Oligomers 40 uL of a 50% aqueous suspension of DEAE-Sepharose was added to a well in a 384-well filter plate. The resin was washed three times with 90 uL of 10 mM acetic acid in water with 0.02% Tween-20, then spun dry in a centrifuge. Purified amino-TEG modified oligomer prepared by Trilink (amino- triethyleneglycol-5'-GCTCGTCGCATTCGGCACGC-3') (SEQ. ID. NO.
- the resin was washed with water (90 uL), 50% dimethylacetamide (DMA)/water (90 uL), and DMA (3 x 90 uL) and spun dry in a centrifuge. 50 uL 20% triethylamine in DMA was added to the well, and allowed to stand at room temperature for 7 minutes.
- MeOH/DMA (3 x 90 uL) and then spun dry in a centrifuge.
- the resin was and then spun dry in a centrifuge, rinsed with 50%
- MeOH/DMA (90 uL) and then spun dry in a centrifuge.
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DE10302421A1 (en) * | 2003-01-21 | 2004-07-29 | Ribopharma Ag | New double-stranded interfering RNA, useful for inhibiting hepatitis C virus, has one strand linked to a lipophilic group to improve activity and eliminate the need for transfection auxiliaries |
EP1935434A1 (en) * | 2006-12-19 | 2008-06-25 | Novosom AG | Construction and use of transfection enhancer elements |
AU2008340355B2 (en) * | 2007-12-04 | 2015-01-22 | Tekmira Pharmaceuticals Corporation | Targeting lipids |
CA2882268A1 (en) * | 2014-02-17 | 2015-08-17 | The Royal Institution For The Advancement Of Learning / Mcgill University | Polynucleotide-poly(diol) conjugates, process of preparation and uses thereof |
-
2016
- 2016-10-10 JP JP2018517731A patent/JP2018535197A/en active Pending
- 2016-10-10 EP EP16854552.3A patent/EP3359640A4/en not_active Withdrawn
- 2016-10-10 WO PCT/US2016/056318 patent/WO2017062973A2/en active Application Filing
- 2016-10-10 US US15/289,946 patent/US20170153257A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
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JP2018535197A (en) | 2018-11-29 |
US20170153257A1 (en) | 2017-06-01 |
WO2017062973A2 (en) | 2017-04-13 |
WO2017062973A3 (en) | 2017-05-26 |
EP3359640A4 (en) | 2019-03-20 |
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